As used herein, the term “angiogenesis” means the generation of new blood vessels into a tissue or organ. Under normal physiological conditions, humans or animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta. However, angiogenesis also occurs under abnormal or undesired conditions such as during tumor development, growth and metastasis. This type of angiogenesis may also be referred to as uncontrolled angiogenesis.
Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and pericytes surrounded by a basement membrane form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a “sprout” off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
Persistent, unregulated angiogenesis occurs in a multiplicity of disease states, tumor metastasis and abnormal growth by endothelial cells. The diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic dependent or angiogenic associated diseases. The hypothesis that tumor growth is angiogenesis-dependent was first proposed in 1971 by M. Judah Folkman. (Folkman J., “Tumor angiogenesis: Therapeutic implications” N. Engl. Jour. Med 285:1182–1186 (1971)). In its simplest terms the hypothesis states: “Once tumor ‘take’ has occurred, every increase in tumor cell population must be preceded by an increase in new capillaries converging on the tumor.” Tumor ‘take’ is currently understood to indicate a pre-vascular phase of tumor growth in which a population of tumor cells occupying a few cubic millimeters volume and not exceeding a few million cells, can survive on existing host microvessels. Expansion of tumor volume beyond this phase requires the induction of new capillary blood vessels.
Several molecules have been discovered that inhibit angiogenesis, inhibit tumor growth, cause regression of primary tumors, and/or inhibit metastasis of primary tumors. These molecules are called antiangiogenic agents. One of these antiangiogenic agents has been termed angiostatin, which is a fragment of a plasminogen protein. Angiostatin was first described in U.S. Pat. No. 5,639,725.
Plasminogen protein comprises five kringle region domains and a serine protease domain located at the carboxy-terminal region. The DNA sequence of human plasminogen has been published. (Browne, M. J., et al., “Expression of recombinant human plasminogen and aglycoplasminogen in HeLa cells” Fibrinolysis 5(4):257–260 (1991)). Each kringle region of the plasminogen molecule contains approximately 80 amino acids and contains 3 disulfide bonds. (Robbins, K. C., “The plasminogen-plasmin enzyme system” Hemostasis and Thrombosis) Basic Principles and Practice, 2nd Edition, ed. by Colman, R. W. et al. J. B. Lippincott Company, pp. 340–357, 1987). The approximate amino acid spans of each kringle domain of human plasminogen are as follows: kringle 1 typically encompasses Cys84–Cysl62, kringle 2 typically encompasses Cysl66–Cys243, kringle 3 typically encompasses Cys256–Cys333, kringle 4 typically encompasses Cys358–Cys435, and kringle 5 typically encompasses Cys462–Cys541. (Castellino, F. J. and McCance, S. G., “The kringle domains of human plasminogen” Ciba Found. Symp., 212:46–65 (1997)). Because each kringle domain is separated by an approximately 20 amino acid inter-kringle domain, a kringle region as herein defined can include any portion of these adjacent inter-kringle domains.
Angiostatin protein comprises one or more of these five kringle regions of plasminogen. Among the proteases suggested to be responsible for angiostatin generation are macrophage metalloelastase, metalloproteinases (mmp's) −3, −7, and −9 and plasmin itself in the presence of a free sulphydryl donor such as cysteine. (Dong, Z. et al. “Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma” Cell, 88:801–810 (1997); Lijnen, H. R. et al., “Generation of an angiostatin-like fragment from plasminogen by stromelysin-1 (MMP-3)” Biochemistry, 37:4699–4702 (1998); Patterson, B. C. and Sang Q. A., “Angiostatin-converting enzyme activities of human matrilysis (MMP-7) and gelatinase B/type IV collagenase (MMP-9)” J. Biol. Chem. 272:28823–28825 (1997)) (Stathakis, P. et al., “Generation of angiostatin by reduction and proteolysis of plasmin” J. Biol. Chem. 272:20641–20645 (1997); Gately, S. et al., “The mechanism of cancer-mediated conversion of plasminogen to that angiogenesis inhibitor angiostatin” Proc. Natl. Acad. Sci. USA 94:10868–10872 (1997)).
In its glycosylated state, human plasminogen protein contains an N-linked carbohydrate chain at amino acid position Asn-289 and two O-linked mucin type carbohydrate chains at amino acid positions Ser-249 and Thr-346. Since angiostatin protein is a fragment of a plasminogen protein, angiostatin protein also contains the above-described carbohydrate chains. The proteolytic digestion of plasminogen, by tPA and uPA to produce plasmin for example, is known to be modulated by the carbohydrate content of plasminogen. Further the carbohydrate chains are known to modulate binding of plasminogen to cell surface receptors. Carbohydrate is also known to play a general role in systemic half life of circulating proteins. (Mori, K. et al., “The activation of type 1 and type 2 plasminogen by type I and type II tissue plasminogen activator” J. Biol. Chem. 270:3261–3267 (1995); Davidson, D. J. and Castellino, F. J., “The influence of the nature of the asparagine 289-linked oligosaccharide on the activation by urokinase and lysine binding properties of natural and recombinant human plasminogens” J. Clin. Invest. 92:249–254 (1993); Edelberg, J. et al. “Neonatal plasminogen displays altered cell surface binding and activation kinetics” J. Clin. Invest. 86:107–112 (1990); Gonzales-Gronow, M. et al. “Further characterization of the cellular plasminogen binding site: evidence that plasminogen 2 and lipoprotein a compete for the same site” Biochemistry, 28:2374–2377 (1989); Jenkins, N. et al. “Getting the glycosylation right: implications for the biotechnology industry” Nat. Biotechnol. 14:975–981 (1996)).
The mechanism underlying how angiostatin and its related kringle fragments specifically inhibit endothelial cell growth remains uncharacterized. It is not yet clear whether the inhibition is mediated by a receptor that is specifically expressed in proliferating endothelial cells, or if angiostatin is internalized by endothelial cells and subsequently inhibits cell proliferation. Alternatively, angiostatin may interact with an endothelial cell adhesion receptor such as integrin avb3, blocking integrin-mediated angiogenesis (Brooks, P. C., et al. “Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels” Cell 79:1157–1164 (1994)).
Although angiostatin has been identified as an angiogenesis inhibitor, what is needed in the art are kringle region fragments of plasminogen that have increased antiangiogenic activity. These improved kringle region proteins should be useful for the treatment of angiogenesis-mediated diseases, such as cancer, and for the modulation of other angiogenic processes, such as wound healing and reproduction. Due to the improved nature of the antiangiogenic kringle region proteins, these proteins should be able to be administered in smaller doses, thus lowering the cost of cancer treatment. What is also needed in the art are compositions and methods for the detection, measurement and localization of improved antiangiogenic kringle region proteins.